Protein tyrosine kinases (PTKs) comprise a large and diverse class of proteins having enzymatic activity. The PTKs play an important role in the control of cell growth and differentiation (for review, see Schlessinger and Ullhrich, 1992, Neuron 9:383-391).
For example, receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), typically followed by receptor dimerization, transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signalling molecules that facilitate the appropriate cellular response. (e.g., cell division, metabolic effects, responses to the extracellular microenvironment) see Schlessinger and Ullhrich, 1992, Neuron 9:1-20.
With respect to receptor tyrosine kinases, it has been shown also that tyrosine phosphorylation sites function as high-affinity binding sites for SH2 (src homology) domains of signaling molecules (Fantl et al., 1992, Cell 69:413-423; Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785; Songyang et al., 1993, Cell 72:767-778; and Koch et al., 1991, Science 252:668-678). Several intracellular substrate proteins that associate with receptor tyrosine kinases (RTKs) have been identified. They may be divided into two principal groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such a domain but serve as adapters and associate with catalytically active molecules (Songyang et al., 1993, Cell 72:767-778). The specificity of the interactions between receptors or proteins and SH2 domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. Differences in the binding affinities between SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors are consistent with the observed differences in their substrate phosphorylation profiles (Songyang et al., 1993, Cell 72:767-778). These observations suggest that the function of each receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability but also by the array of downstream signal transduction pathways that are activated by a particular receptor. Thus, phosphorylation provides an important regulatory step which determines the selectivity of signalling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors.
Aberrant stimulation, expression or mutations in the PTKs have been shown to lead to either uncontrolled cell proliferation (e.g., malignant tumor growth) or to defects in key developmental or reparative processes. Consequently, the biomedical community has expended significant resources to discover the specific biological role of members of the PTK family, their function in differentiation processes, their involvement in tumorigenesis and in other diseases, the biochemical mechanisms underlying their signal transduction pathways activated upon ligand stimulation and the development of novel drugs.
Tyrosine kinases can be of the receptor-type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular).
Receptor Tyrosine Kinases (RTKs). The RTKs comprise a large family of transmembrane receptors with diverse biological activities. The receptor tyrosine kinase (RTK) family includes receptors that are crucial for the growth and differentiation of a variety of cell types (Yarden and Ullrich, Ann. Rev. Biochem. 57:433-478, 1988; Ullrich and Schlessinger, Cell 61:243-254, 1990). The intrinsic function of RTKs is activated upon ligand binding, which results in phosphorylation of the receptor and multiple cellular substrates, and subsequently in a variety of cellular responses (Ullrich and Schlessinger, 1990, Cell 61:203-212).
At present, at least nineteen (19) distinct RTK subfamilies have been identified. One RTK subfamily, designated the HER subfamily, is believed to be comprised of EGFR, HER2, HER3, and HER4. Ligands to the HER subfamily of receptors include epithelial growth factor (EGF), TGF-xcex1, amphiregulin, HB-EGF, betacellulin and heregulin. Aberrant regulation of HER2/erbB2 kinase activity is believed to promote a transformed tumorigenic phenotype especially in breast carcinomas. Two other RTK subfamilies are designated the insulin receptor subfamily, which is comprised of INS-R, IGF-1R and IR-R, and xe2x80x9cPDGFRxe2x80x9d subfamily which includes the PDGFxcex1 and xcex2-receptors, CSF-1R, and c-kit.
Several receptor tyrosine kinases, and growth factors that bind thereto, have been suggested to play a role in angiogenesis, although some may promote angiogenesis indirectly (Mustonen and Alitalo, J. Cell Biol. 129:895-898, 1995). One such receptor tyrosine kinase, known as fetal liver kinase 1 (flk-1), is a member of the type III subclass of RTKs. An alternative designation for human flk-1 is kinase insert domain-containing receptor (KDR) (Terman et al., Oncogene 6:1677-83, 1991). Another alternative designation for flk-1/KDR is xe2x80x9cvascular endothelial cell growth factor receptor 2xe2x80x9d (VEGFR-2). The murine version of flk-1/VEGFR-2 has also been called NYK (Oelrichs et al, Oncogene 8(1):11-15, 1993). DNAs encoding mouse, rat and human flk-1 have been isolated, and the nucleotide and encoded amino acid sequences reported (Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026-30, 1991; Terman et al., 1991, supra; Terman et al., Biochem. Biophys. Res. Comm. 187:1579-86, 1992; Sarzani et al., supra; and Millauer et al., Cell 72:835-846, 1993).
The type III subclass RTK designated fms-like tyrosine kinase-1 (fit-1) is related to flk-1/KDR (DeVries et al. Science 255;989-991, 1992; Shibuya et al., Oncogene 5:519-524, 1990). An alternative designation for flt-1 is xe2x80x9cvascular endothelial cell growth factor receptor 1xe2x80x9d (VEGFR-1). To date, members of the flk-1/KDR/VEGFR-2 and flt-1/VEGFR-1 subfamilies have been found expressed primarily on endothelial cells. These subclass members are specifically stimulated by members of the vascular endothelial cell growth factor (VEGF) family of ligands (Klagsburn and D""Amore, Cytokine and Growth Factor Reviews 7: 259-270, 1996). Flt-1 is believed to be essential for endothelial organization during vascular development. Flt-1 expression is associated with early vascular development in mouse embryos, and with neovascularization during wound healing (Mustonen and Alitalo, supra). Expression of flt-1 in adult organs suggests an additional function for this receptor that is not related to cell growth (Mustonen and Alitalo, supra).
Another RTK that is related to flt-1 and flk-1/KDR is fit-4 (Galland et al., Oncogene 8:1233-40, 1993; Pajusola et al., Oncogene 8:2931-37, 1993). Features shared by these three receptors include the seven immunoglobulin-like domains in their extracellular region. The amino acid sequence of flt-4 exhibits significant homology with the sequences of flt-1 and flk-1/KDR, especially in the tyrosine kinase domain (Galland et al., supra). Unlike flt-1 and flk-1, however, a precursor form of flt-4 is cleaved during post-translational processing to form two disulfide-linked polypeptides (Pajusola et al., supra). Studies of flt-4 expression during development support the theory of venous origin of lymphatic vessels (Kaipainen et al., Proc. Natl. Acad. Sci. USA 92:3566-70, April, 1995).
Given the crucial role of endothelial cells in angiogenesis, growth factors that act on endothelial cells are of particular interest for studies of the regulation of vascularization. One such factor is vascular endothelial cell growth factor (VEGF), which binds to both flk-1/KDR and flt-1 with relatively high affinity and is mitogenic toward vascular endothelial cells (Terman et al., 1992, supra; Mustonen et al. supra; DeVries et al., supra). VEGF does not bind to flt-4 (Pajusola et al., supra). The studies reported in Millauer et al., supra, suggest that VEGF and flk-1/KDR/VEGFR-2 are a ligand-receptor pair that play an important role in the formation and sprouting of blood vessels, termed vasculogenesis and angiogenesis, respectively.
Different forms of VEGF arising from alternative splicing of mRNA have been reported, including the four species described by Ferrara et al. (J. Cell. Biochem. 47:211-218, 1991). Both secreted and predominantly cell-associated species of VEGF were identified by Ferrara et al. supra, and the protein is known to exist in the form of disulfide linked dimers.
It is also known that there are a variety of physiological and biochemical mechanisms that underlie edema and the formation of the edematous state in an individual. An important mediator in one or more of these mechanisms is xe2x80x9cvascular endothelial cell growth factorxe2x80x9d (VEGF). This mediator is also known as xe2x80x9cvascular permeability factorxe2x80x9d (VPF). This factor upregulates transport in vascular endothelial cells, and causes an increase in the permeability of numerous vascular beds including the skin, subcutaneous tissues, peritoneal wall, mesentery, diaphragm, trachea, bronchi, duodenum and uterus. Significant diapedesis, alterations in exchange across the endothelium, extravasation and deposition of macromolecules at these sites and prolonged hypotension may accompany these increased permeability effects. These processes are thought to be a facilitating prelude to neovascularization. VEGF is expressed by inflammatory T-cells, macrophages, neutrophils and eosinophils, etc. at sites of inflammation. This factor is upregulated by hypoxia, certain vasopressor hormones, growth factors, reproductive hormones and numerous inflammatory cytokines. VEGF-mediated vascular permeability has been implicated in such disorders as tumor ascites, endometriosis, edematous responses to burns and trauma, endothelial dysfunction in diabetes, ovarian hyperstimulation syndrome complications, and ocular edema.
Thus, it is apparent that the inhibition of VEGF production or activity would be beneficial, especially to block the manifestation of the above-listed disorders. In particular, agents that are capable of blocking VEGF mediated hyperpermeability and edema and associated syndromes would be useful for alleviating these disorders.
Placenta growth factor (PlGF) has an amino acid sequence that exhibits significant homology to the VEGF sequence (Park et al., J. Biol. Chem. 269:25646-54, 1994; Maglione et al. Oncogene 8:925-31, 1993). As with VEGF, different species of PlGF arise from alternative splicing of mRNA, and the protein exists in dimeric form (Park et al., supra). PlGF binds flt-1 with high affinity, but not flk-1/KDR (Park et al., supra). PlGF potentiates the mitogenic effect of VEGF on endothelial cells when VEGF is present at low concentrations, but has no detectable effect when VEGF is present at higher concentrations (Park et al., supra).
Two other subfamilies of RTKs have been designated as the FGF receptor family (FGFR1, FGFR2, FGFR3 and FGFR4) and the Met subfamily (c-met and Ron).
Because of the similarities between the PDGF and FLK subfamilies, the two subfamilies are often considered together. The known RTK subfamilies are identified in Plowman et al., 1994, DNandP 7(6):334-339, which is incorporated herein by reference.
Inhibiting angiogenesis is desirable in certain clinical situations (e.g., to suppress growth and metastasis of solid tumors, or in treating rheumatoid arthritis), whereas promotion of vascularization is beneficial for treating other conditions (e.g., wound healing). Consequently, molecules that promote angiogenesis by transducing signals through the above-discussed receptors, and molecules capable of inhibiting such signal transduction, are both of interest.
The Non-Receptor Tyrosine Kinases. The non-receptor tyrosine kinases represent a collection of cellular enzymes which lack extracellular and transmembrane sequences. At present, over twenty-four individual non-receptor tyrosine kinases, comprising eleven (11) subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack and LIMK) have been identified. At present, the Src subfamily of non-receptor tyrosine kinases is comprised of the largest number of PTKs and include Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. The Src subfamily of enzymes has been linked to oncogenesis. A more detailed discussion of non-receptor tyrosine kinases is provided in Bolen, 1993, Oncogene 8:2025-2031, which is incorporated herein by reference.
Many of the tyrosine kinases, whether an RTK or non-receptor tyrosine kinase, have been found to be involved in cellular signaling pathways involved in numerous pathogenic conditions, including cancer, psoriasis, and other hyperproliferative disorders or hyper-immune responses.
Development of Compounds to Modulate the PTKs. In view of the surmised importance of PTKs to the control, regulation, and modulation of cell proliferation, the diseases and disorders associated with abnormal cell proliferation, many attempts have been made to identify receptor and non-receptor tyrosine kinase xe2x80x9cinhibitorsxe2x80x9d using a variety of approaches, including the use of mutant ligands (U.S. application Ser. No. 4,966,849), soluble receptors and antibodies (Application No. WO 94/10202; Kendall and Thomas, 1994, Proc. Natl. Acad. Sci 90:10705-09; Kim et al., 1993, Nature 362:841-844), RNA ligands (Jellinek, et al., Biochemistry 33:10450-56; Takano, et al., 1993, Mol. Bio. Cell 4:358A; Kinsella, et al. 1992, Exp. Cell Res. 199:56-62; Wright, et al., 1992, J. Cellular Phys. 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., 1994, Proc. Am. Assoc. Cancer Res. 35:2268).
More recently, attempts have been made to identify small molecules which act as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), and vinylene-azaindole derivatives (PCT WO 94/14808) have been described generally as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 A1), seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer.
The identification of effective small compounds which specifically inhibit tyrosine signal transduction by modulating the activity of receptor and non-receptor tyrosine kinases to regulate and modulate abnormal or inappropriate cell proliferation is therefore desirable.
This invention is directed to compounds of the formula: 
wherein ring A represents the structures: 
and
R1, R2 and R4 and R5 each independently represent hydrogen, halogen having an atomic weight of about 19 to 36, lower alkyl having 1 to 4 carbon atoms, lower alkoxy, trifluoromethyl or
R1, R2 and R4, R3 together independently represent methylenedioxy attached to adjacent carbon atoms;
R3 is hydrogen, COR6, or SO2R6 
R6 is xe2x80x94CH3 or 
wherein Y is hydrogen, chlorine or xe2x80x94CH3.
Enantiomers, tautomers, and mixtures of these compounds are included in this invention. Pharmaceutically acceptable acid addition salts of these compounds are also included in this invention.
The compounds of this invention are useful as inhibitors of the tyrosine kinase activity. In particular, the compounds of this invention are useful as inhibitors of tyrosine kinases that are important in the process of angiogenesis. These compounds are also useful as inhibitors of tyrosine kinases that are important in the process of vascular hyperpermeability. Since these compounds are anti-angiogenic and inhibit vascular hyperpermeability, they are important substances for inhibiting the progression of disease states where angiogenesis and vascular hyperpermeability are important components.
Preferred compounds of the present invention have the formulae: 
The present invention further includes the use of these compounds in pharmaceutical compositions with a pharmaceutically effective amount of the above-described compounds and a pharmaceutically acceptable carrier or excipient. These pharmaceutical compositions can be administered to individuals to slow or halt the process of angiogenesis in angiogenesis-aided diseases or of vascular hyperpermeability in diseases associated with this process.
The compounds of this invention have antiangiogenic properties. For this reason, these compounds can be used as active agents against such disease states as arthritis, atherosclerosis, psoriasis, hemangiomas, myocardial angiogenesis, coronary and cerebral collaterals, ischemic limb angiogenesis, wound healing, peptic ulcer Helicobacter related diseases, fractures, cat scratch fever, rubeosis, neovascular glaucoma and retinopathies such as those associated with diabetic retinopathy or age-related macular degeneration. In addition, some of these compounds can be used as active agents against solid tumors and hyperproliferative disorders since such diseases require a proliferation of blood vessel cells for growth and metastasis.
The compounds of this invention have inhibitory activity against tyrosine kinases. That is, these compounds modulate signal transduction by tyrosine kinases. In particular, these compounds selectively inhibit the activity of the KDR/FLK-1/VEGFR-2 tyrosine kinases. Certain compounds of this invention also inhibit the activity of additional tyrosine kinases such as Flt-1/VEGFR-1, Src-subfamily kinases such as Lck, Src, fyn, yes, etc. The occurrence and potency of the inhibitory activity of the compounds of this invention against a particular tyrosine kinase is dependent on the nature, number and arrangement of the substituents (i.e., R1, R2, R3, R4, R5 and R6) of these compounds.
The compounds of this invention, when administered to individuals in need of such compounds, also inhibit vascular hyperpermeability and the formation of edema in these individuals. These compounds act, it is believed, by inhibiting the activity mediated by VEGF receptors which are involved in the process of vascular hyperpermeability and edema formation. VEGF is unique in that it is the only angiogenic growth factor known to contribute directly to vascular hyperpermeability and the formation of edema. Indeed, vascular hyperpermeability and edema that is associated with the expression or administration of many other growth factors appears to be mediated via VEGF production. Diapedesis also often accompanies vascular hyperpermeability. Similarly, excessive vascular hyperpermeability can disrupt normal molecular exchange across the endothelium in critical tissues and organs (e.g., lung and kidney), thereby perturbing function and causing macromolecular extravasation and deposition. By inhibiting the kinase activity of VEGF receptors, hyperpermeability, as well as associated extravasation, subsequent edema or ascites formation and matrix deposition is inhibited and minimized.
In addition, the N-acylated (R3=xe2x80x94COR6) forms or N-sulfonylated (R3=xe2x80x94SO2R6) analogs of the compounds of this invention can also serve as prodrugs which act as metabolically activated antiangiogenic agents.
It is envisaged that the disorders listed above are mediated to a significant extent by protein tyrosine kinase activity involving the KDR/VEGFR-2 and/or the Flt-1/VEGFR-1 tyrosine kinases. By inhibiting the activity of these tyrosine kinases, the progression of the listed disorders is inhibited because the angiogenic component of the disease state is severely curtailed. The action of the compounds of this invention, by their selectivity for specific tyrosine kinases, result in a minimization of side effects that would occur if less selective tyrosine kinase inhibitors were used.
The potency and specificity of the generic compounds of this invention can often be altered and optimized by substituent variations and conformational restrictions.
In this invention, the following definitions are applicable:
xe2x80x9cPharmaceutically acceptable acid addition saltxe2x80x9d refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid or organic acids such as sulfonic acid, carboxylic acid, organic phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, lactic acid, tartaric acid and the like.
xe2x80x9cAlkylxe2x80x9d refers to a saturated aliphatic hydrocarbon, including straight-chain and branched-chain groups having 1 to 4 carbons.
xe2x80x9cAlkoxyxe2x80x9d refers to an xe2x80x9cO-alkylxe2x80x9d group, where xe2x80x9calkylxe2x80x9d is defined as described above.
The identified compounds of this invention can be administered to a human patient by themselves or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms. Techniques for formulation and administration of the compounds of the instant application may be found in xe2x80x9cRemington""s Pharmaceutical Sciences,xe2x80x9d Mack Publishing Co., Easton, Pa., latest edition.
Suitable routes of administration may, for example, include oral, eyedrop, rectal, transmucosal, topical, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the compound in a local rather than a systemic manner, for example, via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks""s solution, Ringer""s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethysulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the PTK modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protic solvents than are the corresponding free base forms.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cellular assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cellular assays (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PTK activity). Such information can be used to more accurately determine useful doses in humans.
A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient""s condition. (See e.g., Fingl et al., 1975, in xe2x80x9cThe Pharmacological Basis of Therapeuticsxe2x80x9d, Ch. 1 p1).
Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using the MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject""s weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition. Suitable conditions indicated on the label may include treatment of a cellular hyperproliferative disorder, inhibition of angiogenesis, treatment of fibrosis, diabetes, edema, ascites and the like.
There are two general approaches to the synthesis of the indeno-, naphtho-, and cyclohepta[1,2-c]pyrazole ring systems that have been set forth in U.S. Pat. No. 3,843,665 and U.S. Pat. No. 3,843,666 and represented by formula I.
In U.S. Pat. No. 3,843,665, cyclization of the pyrazole ring is affected by heating compounds of the structure II with an aromatic sulfonylhydrazide in an inert solvent and a catalytic amount of an acid. The reaction is carried out for a period of 5 to 30 hours at a temperature of 75 to 100 C. The following scheme is representative of this transformation. 
Wherein:
p is 0, 1, 2; and
W is a lower alkyl.
Compounds of formula II are prepared by treating an appropriately functionalized 1-indanone, alpha-tetralone, or 1-benzosuberone with an arylaldehyde in the presence of an acid or base catalyst (Braun, R. A.; Mosher, W. A. J. Amer. Chem. Soc. 1958, 80, 2749). 
A second method of preparing indeno-, naphtho-, and benzo[6,7]cyclohepta[1,2-c]pyrazole is set forth by U.S. Pat. No. 3,843,666 where compounds with the general formula VI are heated to 75xc2x0 to 175xc2x0 C. with a catalytic amount of an organic carboxylic acid or an organic sulfonic acid in an inert solvent such as an aromatic hydrocarbon for a period of 6 to 24 hours. 
Wherein:
Ar, R3 and R2 are as set forth above.
Compounds which have the general formula of VI are prepared by treating compounds of the general formula VII with hydrazine in an inert solvent. The reaction is carried out at 15xc2x0 to 20xc2x0 C. for a period of up to 24 hours. 
Compounds which have the general formula VII are prepared by treating II with alkaline hydrogen peroxide at ambient temperature in a suitable organic cosolvent mixture such as CH2Cl2/MeOH. Alternatively, VII is produced from the reaction of VIII with an aryl aldehyde under basic conditions. The reaction is carried out in an inert solvent at a temperature between 50xc2x0 and 10xc2x0 C. for a period of 3 to 6 hours. 
Wherein:
Y is any conventional leaving group, such as chlorine, bromine, iodine, tosylate or mesylate.
Cyclization of VI can also be affected by treatment with a mineral acid such as hydrochloric acid, sulfuric acid or phosphoric acid. The reaction is carried out in a lower alkanol at a temperature between 15xc2x0 to 20xc2x0 C. for a period of 12 to 48 hours. The product of the reaction, IX, can then be aromatized to I by heating to a temperature of 50xc2x0 to 150xc2x0 C. with an organic carboxylic acid or an organic sulfonic acid in a straight chain ether or cyclic ether for the period of 8 to 30 hours. 
Compound IX can be diacetylated by treatment with acetic anhydride in an inert solvent such as an aromatic hydrocarbon at a temperature between 35xc2x0 to 200xc2x0 C. for a period of 5 to 8 hours. 
Compound XI can then be aromatized to compound XII by heating to a temperature of 35xc2x0 to 200xc2x0 C. with a mineral acid or an organic acid in an inert solvent for the period of 4 to 8 hours. Finally, compound XII can be converted to I by heating to a temperature of 50xc2x0 to 150xc2x0 C. in an inert solvent such as water or a lower alcohol in the presence of an alkai metal or an alkai metal hydroxide for the period of 8 to 30 hours. 
Compounds with the general formula XIII can be obtained by treating VII with a compound of the formula XIV in the presence of an inert solvent such as a lower alkanol, straight chain ether or cyclic ether at a temperature between 15xc2x0 to 20xc2x0 C. for a period of 24 hours. 
Wherein:
R3 is hydrogen, xe2x80x94COR6, or xe2x80x94SOR6 and
R6 is xe2x80x94CH3 or 
xe2x80x83Y is hydrogen, chlorine or xe2x80x94CH3.
Compounds with the general formula XIII can be cyclized to XV by heating to 75xc2x0 to 175xc2x0 C. with a catalytic amount of an organic carboxylic acid or an organic sulfonic acid in an inert solvent such as an aromatic hydrocarbon for the period of 6 to 24 hours. Compounds with the general formula XV (R3xe2x89xa0H) can be converted to I by heating to a temperature of 50xc2x0 to 150xc2x0 C. in an inert solvent such as water or a lower alcohol in the presence of an alkali metal or an alkali metal hydroxide for the period of 8 to 30 hours. 
Specific examples of the above transformations can be found in U.S. Pat. Nos. 3,843,665 and 3,843,666.